Cd8 pacificblue

Manufactured by Beckman Coulter

Sourced in Canada, United States

The CD8-PacificBlue is a fluorescent antibody reagent designed for the identification and enumeration of CD8+ T cells in flow cytometry applications. It binds specifically to the CD8 surface antigen expressed on a subset of T lymphocytes.

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Lab products found in correlation

Cd45 krome orange, by Beckman Coulter (7 mentions) Cd14 fitc, by Beckman Coulter (5 mentions) Cd3 pc5, by Beckman Coulter (5 mentions) Cd4 apc, by Beckman Coulter (5 mentions) Cd56 pc7, by Beckman Coulter (5 mentions) Versalyse buffer, by Beckman Coulter (4 mentions) Hla dr ecd, by Beckman Coulter (4 mentions) Cd19 apcalexafluor700, by Beckman Coulter (4 mentions) Cd16 apcalexafluor750, by Beckman Coulter (4 mentions) Navios flow cytometer, by Beckman Coulter (4 mentions) Cd138 pe, by Beckman Coulter (4 mentions) Cd4 krome orange, by Beckman Coulter (2 mentions) Tim 3 apc, by BD (1 mentions) Anti muc1, by Santa Cruz Biotechnology (1 mentions) Kaluza flow analysis software, by Beckman Coulter (1 mentions) Gallios flow cytometer, by Beckman Coulter (1 mentions) Cd3 apc, by Beckman Coulter (1 mentions) Ccr6 pe cy7, by BioLegend (1 mentions) Tcrαβ fitc, by BD (1 mentions) Cd25 ecd, by Beckman Coulter (1 mentions)

8 protocols using cd8 pacificblue

Multiparametric Flow Cytometry of CSF and Blood

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CSF samples were collected in polypropylene tubes and were processed within 20 min. Cells were obtained from EDTA blood by erythrocyte lysis using VersaLyse buffer (Beckman Coulter, Krefeld, Germany) following the manufacturer's instructions. Cells were obtained from CSF by centrifugation (15 min, 290g, 4°C) and incubation in VersaLyse buffer. Cells were stained for 30 min at 4°C using the following fluorochrome-conjugated antibodies: CD14-FITC, CD138-PE, HLA-DR-ECD, CD3-PC5.5, CD56-PC7, CD4-APC, CD19-APCAlexafluor700, CD16-APCAlexafluor750, CD8-PacificBlue, and CD45-KromeOrange (all Beckman Coulter). T-cell subpopulations were further analyzed using the following fluorochrome-conjugated antibodies: CD45RA-FITC, CD27-PE, CD3-ECD, CCR7-PC5.5, CD25-PC7, CD56-APC, CD127-APCAlexafluor700, CD62L-APCAlexafluor750 or PD1-APCAlexafluor750, CD8-PacificBlue, and CD4-KromeOrange (obtained from Beckman Coulter or Ebioscience, Frankfurt, Germany). After washing, all samples were analyzed using the Navios™ flow cytometer (Beckman Coulter, Germany). The gating strategy to determine HLA-DR expression on CD4+ and CD8+ T cells and CD4+ and CD8+ T-cell subpopulations are described in Figures S1 and S2.

Grauer O.M., Reichelt D., Grüneberg U., Lohmann H., Schneider-Hohendorf T., Schulte-Mecklenbeck A., Gross C.C., Meuth S.G., Wiendl H, & Husstedt I.W. (2015). Neurocognitive decline in HIV patients is associated with ongoing T-cell activation in the cerebrospinal fluid. Annals of Clinical and Translational Neurology, 2(9), 906-919.

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Multiparametric T Cell Phenotyping

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The following antibodies were used in this study for T cell phenotyping: CD3-PerCP (clone SK7/Cat# 347344), CD25-APC AF700, CD4-Krome Orange (13B8.2/A96417), CD8-Pacific Blue (B9.11/A82791), CD3-APC (Beckman Coulter Inc. Brea, CA), Rat Anti-Mouse IgG1-APC (X56/550874) (BD Biosciences, San Jose, CA). PD-1-Percp Cy7 and TIM3-APC (BD Biosciences, San Jose, CA) were used as markers of T cell exhaustion. MUC1 antigen expression by tumor cells was measured using anti-MUC1, (Santa Cruz Biotechnology. Inc., Dallas, TX). CAR molecules were detected using Goat anti-human F(ab’)2 antibody conjugated with AlexaFluor647 (109–606-097) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Cells were stained with saturating amounts of antibody (~5uL) for 20 min at 4 °C, washed (PBS, Sigma-Alrich, St. Louis, MO), and then acquired on Gallios™ Flow Cytometer (Beckman Coulter Inc., Brea, CA). Analysis was performed using Kaluza® Flow Analysis Software (Beckman Coulter Inc.).

Bajgain P., Tawinwung S., D’Elia L., Sukumaran S., Watanabe N., Hoyos V., Lulla P., Brenner M.K., Leen A.M, & Vera J.F. (2018). CAR T cell therapy for breast cancer: harnessing the tumor milieu to drive T cell activation. Journal for Immunotherapy of Cancer, 6, 34.

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Flow Cytometric Immune Cell Profiling

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CSF and EDTA blood samples were obtained from all patients and processed as described previously (19 (link)). Briefly, cells from CSF or 100 μl EDTA blood were incubated in VersaLyse buffer (Beckman Coulter; Brea, CA) for 10 min and subsequently washed three times with PBS supplemented with 2% heat-inactivated FCS and 2 mM EDTA. Following incubation with fluorochrome-conjugated antibodies (CD14-FITC, CD138-PE, HLA-DR-ECD, CD3-PC5.5, CD56-PC7, CD4-APC, CD19-APCAlexafluor700, CD16-APCAlexafluor750, CD8-PacificBlue, and CD45-KromeOrange, all Beckman Coulter), cells were acquired on a Navios flow cytometer (Beckman Coulter). Analysis was conducted with Kaluza V1.2. Lymphocytes, monocytes, and granulocytes were selected based on forwards scatter channel, sideward scatter channel, CD14, and CD45 expression characteristics. Lymphocytes subsets were selected as CD3⁺CD4⁺ (T helper cells), CD3⁺CD8⁺ (cytotoxic T cells), CD3⁺HLA-DR⁺ (activated T cells), CD3⁻CD56⁺ (NK cells), CD3⁻CD19⁺ (B cells), CD3⁻CD19⁺CD138⁺ (plasma cells), whereas monocyte subsets were selected as CD14⁺CD16⁻ (classical monocytes), CD14⁺CD16⁺ (non-classical monocytes) cells.

Ruland T., Wolbert J., Gottschalk M.G., König S., Schulte-Mecklenbeck A., Minnerup J., Meuth S.G., Groß C.C., Wiendl H, & Meyer zu Hörste G. (2018). Cerebrospinal Fluid Concentrations of Neuronal Proteins Are Reduced in Primary Angiitis of the Central Nervous System. Frontiers in Neurology, 9, 407.

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Multiparametric T Cell Immunophenotyping

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Cell surface markers were chosen based on their ability to discriminate subsets in the T cell population of humans. The panel consists of the following antibodies: TCRαβ FITC (Becton Dickinson, Franklin Lakes, NJ), TCRγδ PE (Becton Dickinson, Franklin Lakes, NJ), CD25 ECD (IOTest, Beckman Coulter, Brea, CA), CD4 PERCP (Becton Dickinson, Franklin Lakes, NJ), CCR6 PE-Cy7 (Biolegend, San Diego, CA), CD45RO APC (Becton Dickinson, Franklin Lakes, NJ), IL-23R AF700 (R&D Systems, Minneapolis, MN), CD3 APC-AF750 (IOTest, Beckman Coulter, Brea, CA), CD8 Pacific Blue (IOTest, Beckman Coulter, Brea, CA), or CD45 Krome Orange (IOTest, Beckman Coulter, Brea, CA), corresponding to fluorescent channels FL1-FL10, respectively, for the generation of a compensation matrix.

Byrd T., Carr K.D., Norman J.C., Huye L., Hegde M, & Ahmed N. (2015). Polystyrene microspheres enable 10‐color compensation for immunophenotyping of primary human leukocytes. Cytometry, 87(11), 1038-1046.

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Multiparameter Flow Cytometry for Immune Cells

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Multiparameter flow cytometry of immune cells in PB and CSF samples was done as described previously (24 (link), 29 (link)). During lumbar puncture CSF was sampled into polypropylene tubes. All CSF samples were processed in < 20 min. Cells were isolated from CSF by centrifugation (15 min, 290 g, 4°C) and subsequent incubation in VersaLyse buffer (Beckman Coulter, Germany). PB samples were collected in EDTA monovettes and cells were isolated by using VersaLyse buffer. For immunostainings, the following fluorochrome-conjugated antibodies were used: CD14-FITC, CD138-PE, HLA-DR-ECD, CD3-PC5.5, CD56-PC7, CD4-APC, CD19-APC-Alexafluor700, CD16-APC-Alexafluor750, CD8-PacificBlue, and CD45-KromeOrange (all from Beckman-Coulter). Data acquisition was performed with a Navios flow cytometer (Beckman-Coulter). Gating strategy for Leukocytes and Monocytes is described and illustrated in Supplementary Figure 1.

Schröder J.B., Pawlowski M., Meyer zu Hörste G., Gross C.C., Wiendl H., Meuth S.G., Ruck T, & Warnecke T. (2018). Immune Cell Activation in the Cerebrospinal Fluid of Patients With Parkinson's Disease. Frontiers in Neurology, 9, 1081.

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Immunophenotyping of Peripheral Blood and CSF Cells

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Single-cell suspensions from human peripheral blood mononuclear cells and CSF cells were stained for 30 min at 4 °C with the appropriate combination of indicated fluorescence-labeled monoclonal antibodies in PBS, containing 0.1% sodium azide and 0.1% bovine serum albumin (BSA) following treatment with VersaLyse (Beckman Coulter GmbH, Krefeld, Germany) according to the manufacturer’s instructions. The following monoclonal antibodies were used at 1:200 dilutions: CD14-FITC, CD138-PE, HLA-DR-ECD, CD3-PC5.5, CD56-PC7, CD4-APC, CD19-APCAlexafluor700, CD16-APCAlexafluor750, CD8-PacificBlue, and CD45-KromeOrange (all Beckman Coulter). Flow cytometric analysis of stained cells was performed following standard protocols. Cells were analyzed on a Navios™ flow cytometer (Beckman Coulter) using Kaluza Analysis Software (V2.1, Beckman Coulter) and presented using Prism 6.0 (Graph Pad).

Gross C.C., Pawlitzki M., Schulte-Mecklenbeck A., Rolfes L., Ruck T., Hundehege P., Wiendl H., Herty M, & Meuth S.G. (2020). Generation of a Model to Predict Differentiation and Migration of Lymphocyte Subsets under Homeostatic and CNS Autoinflammatory Conditions. International Journal of Molecular Sciences, 21(6), 2046.

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Comprehensive Immunological Profiling

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Laboratory analyses included C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), total serum IgE, total serum immunoglobulin G (IgG), IgG1, IgG2, IgG3, and IgG4 subclasses. Flow cytometry was performed using a Navios cytometer (Beckman Coulter, Brea, CA, USA) on fresh peripheral blood collected in ethylenediaminetetraacetic acid tubes using a lyse-no-wash technique (ammonium chloride) and the following panel of directly conjugated antibodies: CD3-fluorescein isothiocyanate, CD4-ECD, CD8-Pacific Blue, CD19-A700, CD20-allophycocyanin, CD27-phycoerythrin-cyanine 7, CD38-A750, CD45-Krome Orange, CD56-phycoerythrin, CD138-PC5.5 (Beckman Coulter). Naive B cells, memory B cells, plasmablasts, and plasma cells were identified within the CD19⁺ gate as CD19⁺CD20⁺CD27⁻CD38⁺ cells, CD19⁺CD20⁺CD27⁺CD38⁻ cells, CD19⁺CD20⁻CD27⁺CD38^+bright cells, and CD19⁺CD20⁻CD38⁺CD138⁺ cells, respectively. Total B cells were identified both as CD19⁺ cells (CD19⁺/side scatter [SSC] within the leukogate) and CD20⁺ cells (CD20⁺/SSC within the leukogate).

Lanzillotta M., Della-Torre E., Milani R., Bozzolo E., Bozzalla-Cassione E., Rovati L., Arcidiacono P.G., Partelli S., Falconi M., Ciceri F, & Dagna L. (2018). Increase of circulating memory B cells after glucocorticoid-induced remission identifies patients at risk of IgG4-related disease relapse. Arthritis Research & Therapy, 20, 222.

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CSF Immune Cell Profiling in Rapid GCA

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Patients with rapid progressive GCA were compared to a group of 12 age-matched patients retrospectively diagnosed with somatoform disorders without any signs of inflammatory CSF conditions (ie, <5 cells/μl CSF, <2 mmol/l lactate, no blood/CSF-barrier disruption, no intrathecal immunoglobulin synthesis [Reiber/OCB]). CSF samples were analyzed within 1 hour after lumbar puncture by centrifugation at 290 g for 15 minutes at 4 C in parallel with 100 μl peripheral blood. Blood-tinged CSF samples were excluded from the study. After treatment with VersaLyse buffer (Beckman Coulter) for 10 minutes, the samples were washed twice by addition of 3 ml FC-buffer (PBS [Sigma] supplemented with 2% heat-inactivated FCS Gold [BioSell] and 2 mM EDTA [Sigma]) and subsequent centrifugation at 290 g for 4 minutes. Following staining with CD14-FITC, CD3-PC5.5, CD56-PC7, CD4-APC, CD19-APC-AF700, CD8-PacificBlue, and CD45-KromeOrange (all Beckman Coulter), samples were washed once with FC buffer. After aspirating the supernatant, samples were resuspended and 20 μl flow count fluorospheres (Beckman Coulter) were added prior to acquisition using a Navios flow cytometer (Beckman Coulter). Resulting files were analyzed by Kaluza 2.1 (Beckman Coulter).

Beuker C., Wankner M.C., Thomas C., Strecker J.K., Schmidt-Pogoda A., Schwindt W., Schulte-Mecklenbeck A., Gross C., Wiendl H., Barth P.J., Eckert B., Meinel T.R., Arnold M., Schaumberg J., Krüger S., Deb-Chatterji M., Magnus T., Röther J, & Minnerup J. (2021). Characterization of Extracranial Giant Cell Arteritis with Intracranial Involvement and its Rapidly Progressive Subtype. Annals of neurology, 90(1).

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